Our long-term goal is to study structures, functions, and regulatory mechanisms of vacuolar H+-ATPases (V-ATPases) from the yeast Saccharomyces cerevisiae. In particular, the research is designed to elucidate the role of V-ATPase in cytosolic pH homeostasis and to determine the role of the V[1] subunit E in V-ATPase function. V-ATPases are highly conserved ATP-driven proton pumps distributed among the endomembrane system of all eukaryotic cells and the plasma membrane of certain specialized cells. V-ATPase proton-transport accomplishes diverse roles, from constitutive vacuole and lysosome acidification to maintenance of acid-base balance in humans. The enzyme is a multisubunit complex consisting of two domains; a peripheral domain or V[1], and a membrane domain or V[0]. V[1] and V[0] interactions are essential for V-ATPase function and only assembled V[1]V[0] hydrolyzes ATP and pumps protons across the membrane. Deprivation of glucose, the primary fuel for most cells, reversibly disassembles V[1] from V[0] to down-regulate V-ATPase activity. Glucose-triggered disassembly prevents energy depletion when glucose levels are low. Specific aim #1 of this study (Analysis of Subunit E Phosphorylation and Function by Site-directed Mutagenesis) is designed to determine the impact that phosphorylation of the V[1] subunit E has on V-ATPase functions and reversible disassembly. Specific Aim #2 (Measurement of Cytosolic pH and Nucleotides in vma Mutants) will contribute significantly to the understanding of the physiological mechanisms V-ATPases use to maintain cytosolic pH homeostasis. This aim also endeavors to unravel the basis for the pH- and calcium-sensitive phenotypes vma mutants exhibit. We will exploit yeast for its resemblance other eukaryotic cells and its suitability for biochemistry, cellular, and molecular biology.